During flowering plant reproduction, pollen lands on the surface of the pistil, extends a tube that carries sperm to an ovule and effects fertilization. Pistil cells are critical for inducing pollen germination and tube growth, yet only a few of these signals have been identified. After a pollen grain lands on the surface of a pistil, it absorbs water from the stigma, forms a pollen tube and transports all cellular contents along with both sperm cells to its tip (Weterings and Russell, Plant Cell, 16 (Suppl):S107-118, 2004). Pollen tubes invade the pistil and migrate past several different cell types, growing between the walls of the stigma cells, traveling through the extracellular matrix of the transmitting tissue, and finally arriving at the ovary, where they migrate up the funiculus, and enter the micropyle to fertilize an egg and a central cell (Lord and Russell, Ann. Rev. Cell Dev. Biol., 18:81-105, 2002). Typically, only one pollen tube enters the ovule through the micropyle, terminates its journey within a synergid cell, and bursts to release the two sperm cells, of which one will fuse with the egg cell to generate an embryo and the second one fuses with the central cell to form an endosperm.
A combination of genetic, biochemical and in vitro assays have defined signals that contribute to the early stages of pollen tube guidance. The early phase of pollen tube growth—germination and penetration of the stigma—is influenced by the stigma exudate in plants with wet stigmas (e.g. tobacco) and by the pollen coat in species with dry stigmas (e.g. Arabidopsis). Lipids in the exudate and the pollen coat are required for pollen germination (Preuss et al., Genes Dev, 7:974-985, 1993; and Wolters-Arts et al., Nature, 392:818-821, 1998), perhaps functioning by controlling the flow of water to pollen (Lush, Trends Plant Sci, 4:413-418, 1999).
Besides genetic analyses, in vitro growth assays have been used to characterize pollen tube behavior. Such assays were used to investigate intracellular responses within pollen tubes. For example, cues such as a Ca2+ gradient at the tip of pollen tubes were shown to be critical for their growth. Disrupting this gradient by iontophoretic microinjection or by incubation with Ca2+ channel blockers can change the direction of tube growth (Gilroy and Trewavas, Nat Rev Mol Cell Biol, 2:307-314, 2001). The Ca2+ gradient in pollen tubes is controlled by Rho GTPases; injection of antibodies against these proteins into pollen tubes or expression of dominant-negative forms of Rho GTPase causes the tip-focused Ca2+ gradient to diffuse and eliminates tube growth (Zheng and Yang, Trends Plant Sci, 5:298-303, 2000), presumably by disrupting F-actin assembly (Gu et al., J Cell Biol, 169:127-138, 2005).
In vitro-grown pollen tubes also respond to extracellular growth and guidance cues. Lily pollen tubes are attracted to chemocyanin and repelled by a point source of nitric oxide, NO (Prado et al., Development, 131:2707-2714, 2004). In addition, in vitro grown pearl millet pollen tubes are attracted to ovary extracts (Reger et al., Sexual Plant Reproduction, 5:47-56, 1992). In Torenia fournieri, pollen tube guidance across a simple medium and into the ovule was achieved only after pollen tubes were grown through a stigma and style (Higashiyama et al., Plant Cell, 10:2019-2032, 1998). In this species, the female gametophyte protrudes from the ovule, and pollen tubes enter the micropyle without interacting with a funiculus (Id.). Using this system, it was demonstrated that when synergid cells were ablated, pollen tubes did not penetrate the micropyle, demonstrating that the synergid is the source of the attractant that facilitates pollen tube entry into the micropyle (Higashiyama et al., Science, 293:1480-1483, 2001).
Chrysanthemum in vitro pollen germination was notably increased in the presence of floral organs and although the factor responsible was not identified, it was found to be soluble in water, ether or methanol (Tsukamoto and Matsubara, Plant & Cell Physiol., 9:237-245, 1967). From Petunia stigma extracts, kaempferol was isolated and showed to stimulate pollen germination (Mo et al., PNAS, 89:7213-7217, 1992). Consistent with these observations, loss of maize and Petunia chalcone synthase (chs), the first enzyme of flavonoid biosynthesis, resulted in white pollen that was incapable of germinating, growing and fertilizing ovules (Id.). Notwithstanding these observations concerning the role of flavonoids in pollen function, null mutation in the single chalcone synthase gene in Arabidopsis did not affect pollen germination, growth or its ability to fertilize ovules (Burbulis et al., Plant Cell, 8:1013-1025, 1996). Arabidopsis thaliana is arguably the most widely used plant model organism to study plant biology. Thus, a continuing need exists to identify the factors responsible for stimulation of pollen tube germination and growth.